Method for simultaneous detection of reduced and oxidized biothiols

Abstract

The application provides a method for simultaneously detecting reduced and oxidized biological aminothiol compounds, which comprises the following steps: subjecting a sample to be detected to derivatization of two reagents of a sulfydryl protection reagent and [d0] / [d3]-dimethoxymethyl benzisopyrylium salt in sequence to obtain a derivatized sample, and then performing liquid chromatography-mass spectrometry detection on the derivatized sample to obtain a detection result. The biological aminothiol compounds (BATs) are analyzed by using the method, the sample pretreatment step is simple, the conversion of reduced BATs into oxidized BATs is blocked, and qualitative and quantitative analysis of the reduced BATs and the oxidized BATs can be simultaneously realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of analysis, and particularly relates to an analysis method for simultaneously detecting reduced and oxidized biological amino thiol compounds in a biological sample based on a two-step derivatization method. BACKGROUND

[0002] Functional groups of thiol (-SH) and amino (-NH2) are two indispensable functional groups in organisms, wherein the reactivity of thiol is high and easy to be oxidized to form disulfide bonds. A large number of compounds containing both amino and thiol exist in human body, animals, plants and microorganisms. The most common biological amino thiol compounds (BATs) mainly include cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and cysteinyl glycine (CG) and the like. Due to the instability of thiol, the reduced BATs are usually converted into oxidized BATs, such as Cys into cystine (CysSS), Hcy into homocystine (HcySS) and GSH into oxidized glutathione (GSSG). These BATs participate in a series of physiological metabolic processes in organisms, such as GSH has strong reducing property and can remove excessive free radicals in organisms, and has the effects of resisting oxidative stress and maintaining redox balance; the content of Hcy is considered as an index for measuring cardiovascular diseases; Cys in plants has good detoxification effect on heavy metals. In addition, the change of the content of BATs is also proved to be related to a variety of diseases, such as cancer, cardiovascular disease, Alzheimer's disease and diabetes and the like. Therefore, it has important research significance to establish a detection method with high selectivity and high sensitivity for biological thiol compounds.

[0003] Based on different detection techniques, many methods have been developed and applied to the detection analysis of BATs, including colorimetric method, fluorescence method, capillary electrophoresis, electrochemical analysis, liquid chromatography and liquid chromatography-mass spectrometry (LC-MS), but it is still a challenge to achieve reliable BATs analysis. Because BATs are characterized by large polarity, small molecular weight and lack of chromogenic groups, and the reduced form can spontaneously convert to the oxidized form, which increases the difficulty and inaccuracy of BATs analysis. In order to solve the problems caused by the structure of BATs, many derivatization methods have been developed. For example, the introduction of fluorescence or chromophore can facilitate the detection of BATs, but due to structural similarity and similar molecular weight, it is difficult to accurately identify all BATs during detection. In LC-MS detection, the use of derivatization reagents to modify the structure of BATs can prolong the retention time of BATs and improve the MS signal and stability. However, so far, there is no report on simultaneous qualitative and quantitative analysis of reduced and oxidized BATs.

[0004] In view of the various problems existing in the LC-MS analysis method of BATs at present, how to provide a more stable and accurate detection method capable of simultaneously analyzing reduced and oxidized BATs has become a problem to be solved. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a method for simultaneously detecting reduced and oxidized biological aminothiol compounds in a biological sample based on a two-step derivatization method. After the two-step derivatization of BATs with N,N-dimethyl-bromoacetamide (Br-DMA) and [d0]- / [d3]-dimethoxymethylbenzisopyrylium salt ([d0]- / [d3]-DMMIC), liquid chromatography-quadrupole / time-of-flight tandem mass spectrometry (LC-Q-TOF MS) is used for detection.

[0006] The principle of the present application is as follows:

[0007]

[0008] To achieve the purpose of the present application, the following technical solutions are adopted:

[0009] A method for simultaneously detecting reduced and oxidized biological aminothiol compounds, which comprises the steps of: derivatizing the sample to be tested with a thiol protection reagent and a [d0]- / [d3]-dimethoxymethylbenzisopyrylium salt to obtain a derivatized sample, and then detecting the derivatized sample by liquid chromatography-mass spectrometry to obtain a detection result.

[0010] As a preferred embodiment of the present application, the sample to be tested comprises BATs extracted from a biological sample after pretreatment of the biological sample.

[0011] As a preferred embodiment of the present application, the pretreatment step comprises treating the biological sample to be tested with a mixed solution of methanol and water containing Br-DMA (preferably 100 mM) and dichlorophenylalanine (preferably 10 μM), preferably in a volume ratio of 4:1, to extract the BATs in the biological sample.

[0012] As a preferred embodiment of the present application, the BATs include Cys, Hcy, GSH, CG, CysSS, HcySS and GSSG.

[0013] As a preferred embodiment of the present application, the thiol-protecting reagent is Br-DMA, which has the structure shown in Formula I:

[0014]

[0015] The structure of the [d0] / [d3]-dimethoxymethylbenzisoxazolium salt is shown in Formula II:

[0016]

[0017] wherein X is independently selected from H or D.

[0018] As a preferred embodiment of the present application, it specifically comprises the following steps:

[0019] (1) First derivatization reaction: the sample to be tested is reacted with a Br-DMA solution in the presence of a catalyst to generate a derivative product;

[0020] (2) Second derivatization reaction: the sample to be tested is reacted with a [d0] / [d3]-dimethoxymethylbenzisoxazolium salt solution in the presence of a catalyst to generate a post-derivatization sample;

[0021] (3) Liquid chromatography-mass spectrometry detection: the post-derivatization sample is detected by liquid chromatography-quadrupole / time-of-flight tandem mass spectrometry for qualitative and quantitative analysis of aminothiol compounds.

[0022] As a preferred embodiment of the present application, the Br-DMA solution in step (1) includes a Br-DMA solution in acetone, a methanol solution, an aqueous solution, etc. Preferably, the Br-DMA solution is prepared in acetone with a concentration of 100 mM;

[0023] and / or, the molar ratio of the sample to be tested to Br-DMA is 1:10-100; the content of Br-DMA is far in excess of the sample to be tested to ensure sufficient reaction of the BATs in the sample to be tested;

[0024] and / or, the catalyst is an imidazole solution, pyridine or aqueous ammonia, preferably the catalyst is an imidazole solution prepared in ultrapure water with a concentration of 1 M;

[0025] and / or, the reaction temperature is room temperature, and the reaction time is 5-10 s.

[0026] As a preferred embodiment of the present application, the solution of [d0]- / [d3]-dimethoxymethyl benzisoquinolinium salt in step (2) comprises an acetonitrile solution of [d0]- / [d3]-dimethoxymethyl benzisoquinolinium salt, a dichloromethane solution, preferably, the [d0]- / [d3]-DMMIC solution is configured by acetonitrile, and the concentration is 250 mM;

[0027] and / or, the molar ratio of the sample to be tested to [d0]- / [d3]-dimethoxymethyl benzisoquinolinium salt is 1:50-200; the content of [d0]- / [d3]-dimethoxymethyl benzisoquinolinium salt is far more than that of the sample to be tested to meet the sufficient reaction of BATs in the sample to be tested;

[0028] and / or, the catalyst is imidazole, pyridine, etc. Preferably, the catalyst is an imidazole solution configured by ultrapure water, and the concentration is 1 M;

[0029] and / or, the reaction temperature is 35-45℃, and the reaction time is 45-75 min, preferably, the reaction temperature is 40℃, and the reaction time is 1 h.

[0030] As a preferred embodiment of the present application, the specific method of step (3) comprises:

[0031] Agilent 1290 high performance liquid chromatograph-6545 quadrupole time-of-flight mass spectrometer is used;

[0032] and / or, the chromatographic column is Waters XSelect HSS T3 column (2.1x150 mm, 2.5 μm);

[0033] and / or, the spray gas pressure is 20 psi, the atomization gas temperature is 325℃, and the voltage is 2500 V;

[0034] and / or, the mobile phase A is water, and the mobile phase B is methanol;

[0035] and / or, the elution gradient is 10% of the mobile phase B at 0-1 min, the mobile phase B is linearly increased to 30% at 1-2 min, the mobile phase B is linearly increased to 35% at 2-7 min, the mobile phase B is linearly increased to 95% at 7-10 min, the mobile phase B is 95% at 10-13.5 min, the mobile phase B is linearly decreased to 10% at 13.5-14 min, and the mobile phase B is 10% at 14-19 min; the flow rate is 0.3 mL / min;

[0036] and / or, the injection amount is 1 μL.

[0037] As a preferred embodiment of the present application, the reaction of step (2) is also subjected to impurity removal, and the specific method comprises: blowing the reaction solution dry with nitrogen, redissolving the residue in ultrapure water containing 0.3% HCl, and removing impurities by extraction with dichloromethane.

[0038] The present application screens a suitable thiol protection reagent N,N-dimethyl-bromoacetamide (Br-DMA) as a derivatization reagent for the first step of derivatization reaction, and simultaneously screens the optimal reaction conditions, so that the first step of derivatization reaction is realized at room temperature and only needs to react for 5-10 s, the derivatization speed is fast, and the derivatization condition is simple.

[0039] The structure of the Br-DMA reagent is shown in formula I:

[0040]

[0041] The above specific structure of Br-DMA can quickly and effectively react with the thiol group in BATs under the condition of an alkaline catalyst, and the derivatization can be completed in 5-10 s, and the product is stable.

[0042] The reaction principle is as follows:

[0043]

[0044] wherein R-SH is a thiol-containing organic compound.

[0045] On the other hand, the present application screens the optimal reaction conditions for the second step of derivatization reaction.

[0046] The structure of the [d0]- / [d3]-dimethoxymethylbenzisoxazolium salt ([d0]- / [d3]-DMMIC) reagent is shown in formula II:

[0047]

[0048] wherein X is independently selected from H or D.

[0049] When X is H, the compound is denoted as [d0]-DMMIC.

[0050] When X is D, the compound is denoted as [d3]-DMMIC.

[0051] The above specific structure of DMMIC can quickly and effectively react with the amino group in BATs, complete derivatization, and the product can obtain stable mass spectrometry response with high sensitivity, thereby improving the mass spectrometry detection performance of BATs.

[0052] The reaction principle is as follows:

[0053]

[0054] wherein R-NH2 is an amino-containing organic compound.

[0055] Compared with the prior art, the present application has the following beneficial effects:

[0056] The present application provides a two-step derivatization method for BATs and its application in BATs detection. By reacting the derivatization reagent Br-DMA with the sample to be tested, the reduced form of BATs is quickly protected, and the conversion of the reduced form of BATs to the oxidized form of BATs is blocked. Then by reacting the specific structure of [d0]- / [d3]-DMMIC with the sample to be tested, the product can obtain stable high sensitivity mass spectrum response, thereby improving the accuracy and stability of BATs detection.

[0057] The two-step derivatization method established by the present application can quickly block the conversion of the reduced form of BATs to the oxidized form of BATs, and realize accurate qualitative and quantitative analysis by combining the isotopic derivatization reagent. After methodological evaluation, the present application has good linearity (R 2 > 0.99), the minimum detection limit (LOD) and the minimum quantitative limit (LOQ) are between 0.001-0.1 μM, the accuracy is between 79.29-122.7%, the inter-day precision and the intra-day precision are both within 20%, the matrix effect is within the range of 92.69-126.79%, and the recovery rate is within the range of 70.09-119.27%. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 is the liquid chromatography-mass spectrometry detection result diagram of the product of the seven BATs after two-step derivatization by Br-DMA and [d0]-DMMIC;

[0059] Figure 2 is the selected ion chromatogram of the derivatized product of GSH after two-step derivatization by Br-DMA and [d0]- / [d3]-DMMIC;

[0060] Figure 3 is the primary mass spectrum diagram of the derivatized product of GSH after two-step derivatization by Br-DMA and [d0]- / [d3]-DMMIC;

[0061] Figure 4 is the selected ion chromatogram of the derivatized product of GSSG after two-step derivatization by Br-DMA and [d0]- / [d3]-DMMIC;

[0062] Figure 5 is the primary mass spectrum diagram of the derivatized product of GSSG after two-step derivatization by Br-DMA and [d0]- / [d3]-DMMIC;

[0063] Figure 6is the secondary mass spectrum of the derivatization product of GSH by two-step derivatization of Br-DMA and [d0]- / [d3]-DMMIC;

[0064] Figure 7 is the secondary mass spectrum of the derivatization product of GSSG by two-step derivatization of Br-DMA and [d0]- / [d3]-DMMIC;

[0065] Figure 8 is the stability result chart of the two-step derivatization product of BATs for 72 hours continuously;

[0066] Figure 9 is the orthogonal PLS-DA chart of 20 pairs of lung adenocarcinoma cancer / cancer-adjacent tissue of 20 patients by two-step derivatization and liquid chromatography-mass spectrometry detection;

[0067] Figure 10 is the box chart of the relative distribution of BATs of 20 pairs of lung adenocarcinoma cancer / cancer-adjacent tissue of 20 patients by two-step derivatization and liquid chromatography-mass spectrometry detection;

[0068] Figure 11 is the box chart of the relative distribution of reduced BATs / oxidized BATs of 20 pairs of lung adenocarcinoma cancer / cancer-adjacent tissue of 20 patients by two-step derivatization and liquid chromatography-mass spectrometry detection. DETAILED DESCRIPTION

[0069] The technical solutions of the present application are further illustrated below by specific embodiments.

[0070]

Preparation of standard solution of BATs

[0071] The standard solutions of Cys, Hcy, GSH and CG are prepared as a mixed standard aqueous solution of reduced BATs with a concentration of 2M; the standard solutions of CysSS, HcySS and GSSG are prepared as a mixed standard aqueous solution of oxidized BATs with a concentration of 100μM.

[0072]

Liquid chromatography-mass spectrometry detection of two-step derivatization product of BATs

[0073] The standard solution of BATs is prepared as a mixed standard aqueous solution of BATs with a concentration of 50μM.

[0074] The detection instrument and method are: Agilent 1290 high performance liquid-6545 quadrupole time-of-flight mass spectrometer; chromatographic column Waters XSelect HSS T3 column (2.1*150mm, 2.5um); spray gas pressure 20psi, atomizing gas temperature 325℃, voltage 2500V; mobile phase A is water (add 0.4% formic acid), B is methanol; elution gradient is 0-1min, B is 10%, 1-2min, B is linearly increased to 30%, 2-7min, B is linearly increased to 35%, 7-10min, B is linearly increased to 95%, 10-13.5min, B is 95%, 13.5-14min, B is linearly decreased to 10%, 14-19min, B is 10%; flow rate is 0.3mL / min; injection volume is 1ul.

[0075] Take 20ul of the above mixed standard solution, add 80ul of Br-DMA solution (100mM Br-DMA acetone solution) and mix, take 80ul of the mixed solution, add 10ul of imidazole solution (1M), vortex for 5-10s, centrifuge, add 10ul of [d0]- / [d3]-DMMIC (60mM) dissolved in acetonitrile, and react at 40℃ for 1h. Then the reaction solution is blown dry with nitrogen, the residue is redissolved with 80ul of ultrapure water containing 0.3% HC1, and 50ul of dichloromethane is used for extraction. Finally, the redissolved solutions of [d0]- / [d3]-DMMIC derivatization products are mixed in equal volumes, centrifuged, and the supernatant is taken for liquid chromatography-mass spectrometry analysis.

[0076] The selected ion chromatogram of the derivatization product obtained by reacting the mixed standard solution of 7 BATs with Br-DMA and [d0]-DMMIC derivatization reagent is shown in Figure 1 From the figure, it can be found that the derivatization products of the 7 BATs have good chromatographic behavior. Taking GSH as an example, the products derived by Br-DMA and [d0]- / [d3]-DMMIC respectively have the same retention behavior in liquid chromatography, showing the same retention time (see Figure 2 ), and in the first mass spectrum, a group of molecular ion peaks with a difference of 3m / z units is produced (see Figure 3 ); taking GSSG as an example, the products derived by [d0]- / [d3]-DMMIC respectively have the same retention behavior in liquid chromatography, showing the same retention time (see Figure 4 ), and in the first mass spectrum, a group of molecular ion peaks with a difference of 3m / z units is produced (see Figure 5 ).

[0077] As shown in Figure 6As shown, taking GSH as an example, the product after derivatization with Br-DMA and then derivatization with [d0]- / [d3]-DMMIC respectively generates a series of representative fragment ions m / z 118.03 and m / z 204.10 / 207.12 in tandem mass spectrometry; as shown in Figure 7 As shown, taking GSSG as an example, the product after derivatization with [d0]- / [d3]-DMMIC respectively generates a series of representative fragment ions m / z 204.10 / 207.12 in tandem mass spectrometry. This is a regular m / z value difference due to deuterium substitution in the fragment structure.

[0078] The above results show that the detection method provided by the present application, when applied to unknown sample analysis, uses the two-step derivatization of Br-DMA and [d0]- / [d3]-DMMIC combined with liquid chromatography-mass spectrometry detection, uses the above chromatographic characteristics (the same retention time) and mass spectrometric characteristics (regular differences in the m / z values of primary / secondary ions), and can realize the search, identification, qualification and quantification of BATs in the sample to be tested.

[0079] Limit of detection

[0080] The BATs mixed standard stock solution was used to prepare seven BATs mixed standard solutions at different concentration levels (1, 10, 25, 50, 100 and 500 nM) using a Br-DMA solution (100 mM Br-DMA in acetone) as a substrate to investigate the limit of detection.

[0081] 80 μL of the BATs mixed standard solution at the above series of concentration levels was taken, 10 μL of 1 M imidazole solution was added, mixed and centrifuged, 10 μL of 60 mM [d0]-DMMIC solution was added, and the reaction was carried out under the above reaction conditions. After all the sample reactions were completed, the samples were blown dry with nitrogen, the residues were redissolved with 80 μL of ultrapure water containing 0.3% HCl, 50 μL of dichloromethane was added for impurity removal, centrifuged, and detected by liquid chromatography-mass spectrometry (the detection method is the same as above, 3 replicates were prepared for each concentration level, and each sample was detected twice in parallel).

[0082] The results are shown in Table 1. The lowest detection limit (signal-to-noise ratio greater than or equal to 3, and the chromatographic peak contains at least 7 consecutive mass spectra) is between 1-100 nM, and the lowest quantification limit (signal-to-noise ratio greater than or equal to 10, and the chromatographic peak contains at least 15 consecutive mass spectra) is between 1-100 nM.

[0083] Table 1: Detection limit of 7 BATs derivatization products detected by LC-MS

[0084] BATs Limit of detection (nM) Limit of quantification (nM) Cys 1 10 Hcy 1 1 GSH 25 25 CG 25 25 CysSS 100 100 HcySS 50 100 GSSG 50 100

[0085] Linearity and accuracy

[0086] Seven BATs mixed standard solutions at a series of concentration levels were prepared using Br-DMA solution (100 mM Br-DMA in acetone). The concentration levels of Cys and Hcy were 10, 25, 50, 100, and 200 nM; the concentration levels of GSH and CG were 25, 50, 100, 200, and 400 nM; and the concentration levels of CysSS, HcySS, and GSSG were 0.1, 0.2, 0.4, 0.7, and 1.0 μM. The linearity of these solutions was investigated.

[0087] Take 80 μL of the above-mentioned series of BATs mixed standard solutions at various concentration levels, add 10 μL of 1M imidazole solution to each, mix and centrifuge, then add 10 μL of 60 mM [d0]-DMMIC solution, and react under the above reaction conditions. Separately, take 80 μL of a fixed concentration of BATs mixed standard solution (0.2 μM), add 10 μL of 1M imidazole solution, mix and centrifuge, and react with 10 μL of 60 mM [d3]-DMMIC solution as an internal standard for the quantitative method. After all samples have reacted, dry them with nitrogen gas, redissolve the residue in 80 μL of ultrapure water containing 0.3% HCl, extract with 50 μL of dichloromethane to remove impurities, centrifuge, then mix equal volumes of [d0]-derivative and [d3]-derivative, centrifuge, and detect using LC-MS (detection method as above, prepare 3 parallel samples for each concentration level, and detect each sample 3 times in parallel).

[0088] The molecular ion peak [M] of the derivative. + The peak height and the molecular ion peak of the internal standard [M] + A linear regression curve was plotted with the peak area ratio on the Y-axis and the concentration on the X-axis. The results are shown in Table 2. All seven BATs achieved good linear regression, with regression coefficients R0. 2 With a value >0.99 and an accuracy between 79.29% and 122.7%, it shows a good linear correlation.

[0089] Table 27 linearity and accuracy of BATs derivatives detected by LC-MS

[0090]

[0091]

[0092] stability

[0093] The product solution after the preparation of BATs derivatization by Br-DMA and [d0]- / [d3]-DMMIC was prepared (the preparation method is the same as above), and was stored in a 4°C refrigerator. The stability of the derivatization product within 72h was detected by LC-MS (the detection method is the same as above, 3 parallel preparations were prepared for each reagent sample, and each sample was detected 3 times in parallel). Among them, [d0]-derivatization product was used as the internal standard of [d3]-derivatization product. Finally, the ratio of the peak height of the test peak to the peak height of the internal standard was taken as the ordinate, and the storage time was taken as the abscissa to make a stability curve. The results are shown in Figure 8 After 72h storage, the mass spectrum signals of all BATs derivatization products remained stable, indicating that the products after derivatization of the amino thiol compound by Br-DMA and [d0]- / [d3]-DMMIC showed good stability within 72h, which was conducive to the long-term LC-MS analysis of batch samples.

[0094] Intra-day and inter-day precision

[0095] From the above series of concentration levels of 7 BATs mixed standard solutions prepared by Br-DMA solution, low, medium and high concentration levels of BATs mixed standard solution (the concentrations of Cys, Hcy, GSH and CG were 25nM, 100nM, 200nM; the concentrations of CysSS, HcySS and GSSG were 100nM, 400nM, 800nM) were selected, and after the addition of imidazole solution, [d0]-DMMIC was reacted, and [d3]-derivatization product of BATs mixed standard solution with fixed concentration was used as the internal standard (the internal standard concentration is the same as above). After all sample reactions were completed, nitrogen was blown dry, the residue was redissolved with 80μL of ultrapure water containing 0.3% HC1, 50μL of dichloromethane was extracted to remove impurities, centrifuged, and then [d0]-derivatization product and [d3]-derivatization product were mixed in equal volume, centrifuged, and detected by LC-MS (the detection method is the same as above, 3 parallel preparations were prepared for each concentration level sample, and each sample was detected 3 times in parallel). The results are shown in Table 3, the intra-day precision fluctuation range is 5.26-18.78%, and the inter-day precision fluctuation range is 6.52-19.01%, which meets the requirements of quantitative analysis.

[0096] Table 3 Intra-day / inter-day precision of 7 BATs two-step derivatization products detected by LC-MS

[0097]

[0098] Matrix effect

[0099] Take 60 μL of tissue sample solution, nitrogen dry for standby. Take 60 μL of ultrapure water, nitrogen dry for standby. Select low, medium and high concentration levels of BATs mixed standard solution (the concentrations of Cys, Hcy, GSH and CG are 25 nM, 100 nM, 200 nM; the concentrations of CysSS, HcySS and GSSG are 100 nM, 400 nM, 800 nM), after adding imidazole solution, react with [d0]-DMMIC, and use [d3]-derivatized product of fixed concentration BATs mixed standard solution as internal standard (the concentration of internal standard is the same as above). After all sample reactions are completed, dry with nitrogen, redissolve the residue with 80 μL of ultrapure water containing 0.3% HCl, extract impurities with 50 μL of dichloromethane, centrifuge, and add 30 μL of [d0]- / [d3]-derivatized product to the centrifuge tube containing the tissue sample residue or ultrapure water residue. LC-MS detection (detection method is the same as above, 3 replicates for each concentration level, and each sample is detected in triplicate). Matrix effect = peak height of derivatized product in tissue sample residue / peak height of derivatized product in ultrapure water residue x 100%. The results are shown in Table 4, the fluctuation range of matrix effect is 92.69-126.79%, indicating that there is no obvious matrix effect in LC-MS detection, which meets the requirements of quantitative analysis.

[0100] Table 4 Matrix effect of two-step derivatized product of BATs in LC-MS detection

[0101]

[0102] Recovery rate

[0103] Take 60 μL of tissue sample solution, nitrogen dry for standby. Take 80 μL of different concentrations of BATs mixed standard solution (concentrations are 0 nM, 25 nM, 100 nM, 200 nM) and add imidazole solution to react with [d0]-DMMIC, and use [d3]-derivatized product of fixed concentration BATs mixed standard solution as internal standard (the concentration of internal standard is the same as above). After all sample reactions are completed, dry with nitrogen, redissolve the residue with 80 μL of ultrapure water containing 0.3% HCl, extract impurities with 50 μL of dichloromethane, centrifuge, and add 30 μL of [d0]- / [d3]-derivatized product to the centrifuge tube containing the tissue sample residue, and centrifuge. LC-MS detection (detection method is the same as above, 3 replicates for each concentration level, and each sample is detected in triplicate). The standard linear regression method is used to compare the added concentration and actual concentration of each BATs, and the recovery rate is calculated. The results are shown in Table 5, the fluctuation range of recovery rate is 70.09-119.2%, which meets the requirements of quantitative analysis.

[0104] Table 5 Recovery rate of two-step derivatized product of BATs detected by LC-MS

[0105]

[0106] From the above, the two-step derivatization method established by the present application can block the conversion of reduced BATs to oxidized BATs, and combined with isotopic derivatization reagents to achieve accurate qualitative and quantitative analysis. Through the above experiments and methodological evaluation, it is proved that the present application has good linearity (R 2 > 0.99), the minimum detection limit (LOD) and the minimum quantitative limit (LOQ) are between 0.001-0.1 μM, the accuracy is between 79.29-122.7%, the inter-day precision and the intra-day precision are both within 20%, the matrix effect is within the range of 92.69-126.79%, and the recovery rate is within the range of 70.09-119.27%.

[0107] The present application further provides the following specific application examples.

[0108] Application Example 1

[0109] Non-target screening of BATs in lung A549 cells

[0110] The extraction solution is prepared by adding 833.09 mg of Br-DMA solution to 20 μL of dichlorobenzene phenylalanine (5 mM), and then using ultrapure water to dilute the solution to 10 mL, and finally adding 40 mL of methanol.

[0111] The lung A549 cells are counted at 3×10 6 cells / sample, centrifuged, the supernatant is discarded, 100 μL of the extraction solution is added, and the sample is subjected to two rapid freeze-thaw cycles using liquid nitrogen and ultrasonic, centrifuged, 80 μL of the supernatant is taken, 10 μL of imidazole solution is added, and then two aliquots of the cell extraction sample are derivatized using [d0]-DMMIC and [d3]-DMMIC, respectively. After all sample reactions are completed, nitrogen is blown to dryness, the residue is redissolved with 80 μL of ultrapure water containing 0.3% HCl, 50 μL of dichloromethane is used for impurity removal, centrifuged, the [d0]-derivatized product and the [d3]-derivatized product are mixed in equal volumes, centrifuged, and subjected to LC-MS / MS detection. According to the ion pair molecular characteristics of the same RT value and m / z difference of 3.0188±0.02, as well as the characteristic fragments of m / z 118.03 and 204.10, suspected BATs are screened out. Then, the MS 2 spectrum of the suspected BATs is obtained by using the target MS / MS scanning mode, and the structure is identified according to the secondary spectrum information. The results are shown in Table 6. From the lung A549 cells, 16 BATs are identified, of which 11 BATs are successfully identified according to the element composition and the metabolite library matching. The remaining 5 molecular formulas are C7H 14 N2O3S, C9H 18 N2S2, C 10 H 20 N2OS2, C9H 17N3O4S, C 14 H 24 BATs of N4O7S. It is illustrated that the method provided by the present application can effectively identify BATs in unknown biological samples.

[0112] Table 6 Non-target screening results of BATs in lung A549 cells

[0113]

[0114]

[0115] For the above experimental test, since the reduced BATs contain both sulfhydryl and amino structures, if the Br-DMA reagent and the sulfhydryl structure of the reduced BATs are not added, the secondary characteristic fragments for characterizing the sulfhydryl structure will be missing, and when analyzing unknown BATs, it cannot be accurately proved that the unknown compound contains sulfhydryl, and accurate non-target screening cannot be performed.

[0116] Application Example 2

[0117] Differential analysis of BATs in lung adenocarcinoma tissue / cancer adjacent tissue

[0118] Configuration of extraction solution: 833.09 mg of Br-DMA solution is added to 100 μL of dichlorobenzylalanine (5 mM), and the solution is diluted to 50 mL with ultrapure water.

[0119] Take 20 lung adenocarcinoma patients 20 pairs of cancer / cancer adjacent samples (T: cancer; N: adjacent to cancer), wash the surface blood with ice water, absorb the surface moisture with filter paper, weigh, homogenate according to 4 μL extraction solution per mg of tissue sample. The homogenate is centrifuged at 10000 rpm for 3 min, and the supernatant is taken, and 16 μL of methanol is added per mg of tissue sample, and the methanol solution is added to precipitate the protein. Take 80 μL of the supernatant of the cancer / cancer adjacent tissue sample after protein precipitation, add 10 μL of imidazole solution, and react with [d0]-DMMIC according to the above reaction conditions. Mix all the supernatants of the adjacent tissue samples by equal volume as the internal standard tissue sample, and react with [d3]-DMMIC. After all the sample reactions are completed, dry with nitrogen, redissolve the residue in 80 μL of ultrapure water containing 0.3% HC1, extract impurities with 50 μL of dichloromethane, centrifuge, mix [d0]-derivatized products with [d3]-derivatized products by equal volume, centrifuge, and detect by liquid chromatography-mass spectrometry (detection method is the same as above, prepare 3 samples in parallel, and detect 3 times for each sample in parallel). Use the signal ratio of the peak height of the target BATs derivatized product to the peak height of the internal standard BATs derivatized product and the signal ratio of the peak height of the dichlorophenylalanine derivatized product to the peak height of the internal standard dichlorophenylalanine derivatized product, deduct the influence of water in the tissue sample, compare the relative distribution of the target BATs in the cancer / cancer adjacent tissue, and deduct the influence of water in the BATs derivatized product peak height ratio / dichlorophenylalanine derivatized product peak height signal ratio. The results are shown in Table 1. Figures 9-11 As shown in Table 1, there is a certain difference in the distribution of each BATs in the cancer / cancer adjacent tissue of lung adenocarcinoma patients, which shows that the method provided by the present application can detect reduced and oxidized BATs at the same time, and the content distribution of BATs and the content of reduced / oxidized BATs are helpful for distinguishing lung adenocarcinoma cancer tissue / cancer adjacent tissue.

[0120] The above description of the embodiments is for the convenience of those skilled in the art to understand and use the invention. Those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described herein to other embodiments without creative labor. Therefore, the present application is not limited to the above embodiments, and improvements and modifications made by those skilled in the art without departing from the scope of the present application should be within the scope of protection of the present application.

Claims

1. A method for simultaneously detecting reduced and oxidized biological aminothiol compounds, characterized in that, The test sample was derivatized by two reagents, namely a mercapto protecting reagent and [d0]- / [d3]-dimethoxymethylbenzisopyranium salt, to obtain a derivatized sample. The derivatized sample was then detected by liquid chromatography-mass spectrometry to obtain the detection results. The thiol protecting agent is Br-DMA, and its structure is shown in Formula I: Formula I; The structure of the [d0]- / [d3]-dimethoxymethylbenzisopyranium salt is shown in Formula II: Formula II; Where X is independently selected from H or D; When X is all H, the compound is denoted as [d0]-dimethoxymethylbenzoisopyranium salt; When X is always D, the compound is denoted as [d3]-dimethoxymethylbenzoisopyranium salt; Bioactive amino thiol compounds include cysteine, homocysteine, glutathione, cysteine ​​glycine, cystine, homocysteine, and oxidized glutathione; Specifically, the following steps are included: (1) First step of derivatization reaction: The sample to be tested reacts with Br-DMA solution under the action of a catalyst to generate a derivatized product. The reaction temperature is room temperature and the reaction time is 5-10s. The catalyst is imidazole solution, pyridine or ammonia. (2) Second step derivatization reaction: The sample to be tested reacts with the [d0]- / [d3]-dimethoxymethylbenzisopyranium salt solution under the action of a catalyst to generate the derivatized sample. The catalyst is imidazole or pyridine. (3) Liquid chromatography-mass spectrometry detection: Liquid chromatography-quadrupole / time-of-flight tandem mass spectrometry was used to detect the derivatized samples and perform qualitative and quantitative analysis of amino mercapto compounds; After the derivatization is generated in step (2), the sample is further purified. The specific methods include: drying the reaction solution with nitrogen, re-dissolving the residue with ultrapure water containing 0.3% HCl, and extracting with dichloromethane to remove impurities. Step (3) includes the following specific methods: High performance liquid chromatography-quadrupole time-of-flight mass spectrometry was used. The chromatographic column was a Waters XSelect HSS T3 column; The spray pressure is 20 psi, the atomizing gas temperature is 325℃, and the voltage is 2500 V; The mobile phase A is water, and the mobile phase B is methanol; The elution gradient was as follows: phase B was 10% from 0 to 1 min; phase B linearly increased to 30% from 1 to 2 min; phase B linearly increased to 35% from 2 to 7 min; phase B linearly increased to 95% from 7 to 10 min; phase B was 95% from 10 to 13.5 min; phase B linearly decreased to 10% from 13.5 to 14 min; and phase B was 10% from 14 to 19 min. The flow rate was 0.3 mL / min. The injection volume was 1 μL.

2. The method according to claim 1, characterized in that, The test sample includes biological amino thiol compounds extracted from biological samples after pretreatment.

3. The method according to claim 2, characterized in that, The pretreatment steps include treating the biological sample to be tested with a mixed solution of methanol and water containing N,N-dimethyl-bromoacetamide and dichlorophenylalanine to extract biological amino-thiol compounds from the biological sample.

4. The method according to claim 1, characterized in that, In step (1): The Br-DMA solution includes an acetone solution, a methanol solution, and an aqueous solution of Br-DMA; The molar ratio of the sample to be tested to Br-DMA is 1:10-100; The catalyst is an imidazole solution prepared with ultrapure water at a concentration of 1M.

5. The method according to claim 4, characterized in that, The Br-DMA solution was prepared with acetone and had a concentration of 100 mM.

6. The method according to claim 1, characterized in that, The [d0]- / [d3]-dimethoxymethylbenzisopyranium salt solution in step (2) includes an acetonitrile solution and a dichloromethane solution of the [d0]- / [d3]-dimethoxymethylbenzisopyranium salt.

7. The method according to claim 6, characterized in that, In step (2): The [d0]- / [d3]-dimethoxymethylbenzoisopyranium salt solution was prepared with acetonitrile and had a concentration of 250 mM. The molar ratio of the test sample to [d0]- / [d3]-dimethoxymethylbenzoisopyranium salt is 1:50-200; The catalyst is an imidazole solution prepared with ultrapure water at a concentration of 1M. The reaction temperature is 35-45℃, and the reaction time is 45-75 min.

8. The method according to claim 7, characterized in that, In step (2): the reaction temperature is 40°C and the reaction time is 1 hour.

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